Factor VIII also known as antihaemophilic factor A or AHF is a plasma protein that participates in the intrinsic pathway of blood coagulation.
Factor VIII circulates in the blood plasma in an extreme low concentration in the form of a non-covalent complex of two proteins having Factor VIII coagulant activity (Factor VIII:C) and ristocetin co-factor activity (von Willebrand Factor(vWF)), respectively, said complex having a molecular weight of 1-20.times.10.sup.6 D.
Factor VIII:C is absent or defective in individuals suffering from the bleeding disorder Haemophilia A affecting about 5 out of 100,000 persons.
von Willebrand Factor binds to activated platelets in such a manner that the aggregation of the activated platelets is enhanced. This effect may be detected in vitro by aggregation of ristocetin induced platelets. Along with von Willebrands disease a prolonged bleeding time is seen due to the lack of or reduced level of biological activity of von Willebrand Factor.
Haemophiliacs suffering from haemophilia A and patients suffering from severe cases of von Willebrands disease are today treated with concentrates comprising Factor VIII:C/vWF, a treatment which has improved their quality of life and economic capacity considerably and has contributed to an increased length of life for these patients.
Pharmaceutical preparations containing Factor VIII (Factor VIII:C and/or vWF) may be produced from blood or blood plasma. The production of such preparations may be carried out in various known manners, all of which are characterized by low yields of especially Factor VIII:C. Common to nearly all the methods is an initial purification step comprising a cryoprecipitation. By cryoprecipitation frozen plasma is thawed at a temperature of 0.degree.-4.degree. C. which gives rise to a precipitate comprising Factor VIII which may be collected by e.g. centrifugation. Even though the cryoprecipitation is relatively simple it has an essential drawback because when used in larger scale, i.e. pools of plasma comprising more than 5 kg, it gives a low yield of Factor VIII:C (30-45% of the contents of plasma), which means that the final yield is low irrespective of which downstream purification steps are used.
Furthermore, in general a virus inactivating step has been included during the production of Factor VIII preparations. The virus inactivating steps have increased the safety against virus of the preparations considerably but causes in most cases a further reduction of the yield of Factor VIII:C.
The very low overall yields of Factor VIII have lead to a shortage of these preparations on several locations and thus, there is a need of new methods for purifying Factor VIII in high yield to comply with the demand of Factor VIII for the treatment of haemophiliacs.
New methods for isolating Factor VIII directly from plasma in high yield would be of great interest as up to 70% of the contents of Factor VIII in plasma is lost as early as in or before the cryoprecipitation.
Recently it has been tried to isolate Factor VIII directly from plasma using affinity chromatography (Thromb. Haemost., 61,(2), 234-237(1989)) but only a yield of less than 60% and a specific of 1 IU Factor VIII/mg protein of the isolated Factor VIII-containing fraction was obtained.
Gel filtration also named gel permeation chromatography or size exclusion chromatography is a diffusion controlled process that is used for separating solutes according to their size. The solutes are passed through a column packed with inert porous gel particles having a pore size excluding the largest molecules whereas the smaller molecules diffuses into the stationary phase inside the gel particles. Thus, the largest molecules being totally excluded from the gel particles are eluted first with the "void volume", whereas the smaller molecules spend a longer time passing the column and are eluted according to decreasing size with increasing "elution volumes".
Gel filtration may be carried out in two different manners: